Purpose.:
Bestrophin-1 gene (BEST1) mutations are responsible for a broad spectrum of human retinal phenotypes, jointly called bestrophinopathies. Canine multifocal retinopathy (cmr), caused by mutations in the dog gene ortholog, shares numerous phenotypic features with human BEST1-associated disorders. The purpose of this study was the assessment of molecular consequences and pathogenic outcomes of the cmr1 (C73T/R25X) premature termination and the cmr2 (G482A/G161D) missense mutation of the canine model compared with the C87G/Y29X mutation observed in human patients.

Methods.:
Dogs carrying the BEST1 mutation were introduced into a breeding colony and used to produce either carrier or affected offspring. Eyes were collected immediately after euthanatization at the disease-relevant ages and were harvested for expression studies. In parallel, an in vitro cell culture model system was developed and compared with in vivo results.

Results.:
The results demonstrate that cmr1 and human C87G-mutated transcripts bypass the nonsense-mediated mRNA decay machinery, suggesting the AUG proximity effect as an underlying transcriptional mechanism. The truncated protein, however, is not detectable in either species. The in vitro model accurately recapitulates transcriptional and translational expression events observed in vivo and, thus, implies loss of bestrophin-1 function in cmr1-dogs and Y29X-affected patients. Immunofluorescence microscopy of cmr2 mutant showed mislocalization of the protein.

Conclusions.:
Molecular evaluation of cmr mutations in vivo and in vitro constitutes the next step toward elucidating genotype-phenotype interactions concerning human bestrophinopathies and emphasizes the importance of the canine models for studying the complexity of the BEST1 disease mechanism.

Mutations in the BEST1 gene are an important cause of inherited retinal disorders. Hitherto, more than 100 unique allelic variants have been linked to human BEST1 (http://www.hgmd.cf.ac.uk/) and associated with five disease phenotypes, broadly termed bestrophinopathies. Most of these sequence alterations lead to Best disease (also called Best vitelliform macular dystrophy [BVMD]), a juvenile-onset macular degeneration, characterized by abnormal accumulation of lipofuscin-like deposits in the subretinal and sub-RPE spaces and a depressed electro-oculogram light peak.1,2 Additionally, BEST1 mutations are associated with adult-onset foveomacular vitelliform dystrophy,3 autosomal dominant vitreoretinochoroidopathy,4 autosomal recessive bestrophinopathy,5 and, as recently reported, autosomal dominant retinitis pigmentosa.6

Bestrophin-related phenotypes, particularly Best disease, are known for their complexity. Notably, observed pleiotropic effects of BEST1, together with incomplete penetrance and variable clinical expressivity, do not facilitate the dissection of the genotype-phenotype correlation, even within a single family. Consequently, disease progression and severity and the exact impact of the disease on vision impairment are difficult to predict in some BVMD-affected patients. In addition, the juvenile onset of the disease and the limited access to large sample sets make genotype-phenotype analysis an extremely challenging task.

Altered residues define three main mutation hotspots of the bestrophin-1 molecule: the highly evolutionarily conserved N-terminal part of the protein (6–30aa), the second transmembrane domain (80–105aa), and the strongly acidic region in the C terminus, immediately after the last transmembrane domain (293–312aa).7,8 Generally, BEST1 disease-associated mutations consist of substitutions (94%) that include nonsense changes truncating the gene product. Thus far, almost 10% of all known bestrophin-1 mutations have been linked to the multifocal vitelliform dystrophy.9–11 Because these gene alterations have also been described in patients with classic BVMD and adult-onset foveomacular vitelliform dystrophy, the BEST1-related multifocal vitelliform dystrophy is considered a multifocal variant of bestrophinopathies.7

The BEST1 gene product, bestrophin-1, is embedded in the basolateral plasma membrane of the retinal pigment epithelium (RPE).12 Despite considerable controversy, several independent studies have provided persuasive evidence proving that bestrophin-1 is indeed a Ca2+-dependent anion channel participating in epithelial transport across the RPE and demonstrating malfunction caused by BEST1 mutations.8,13–17 However, many aspects of the disease mechanism, including the impact of particular mutations on anion channel dysfunction and its correlation with the observed phenotypic variability, remain ambiguous. Extensive in vitro studies and existing rodent models have significantly contributed to a better understanding of the pathophysiology in BEST1-related retinopathies.18–22 However, because the laboratory-induced mouse and rat models do not replicate the human disease phenotype, it is questionable whether they could contribute to further studies on BEST1 disease etiology or genotype-phenotype interactions or assist in the development of effective treatment strategies.

We previously described three spontaneous bestrophin-1 mutations resulting in canine multifocal retinopathy (cmr), an early-onset, autosomal recessive retinal disorder recognized in several dog breeds.23,24 The distinct sequence alterations in canine BEST1 (cBEST1) include a nonsense transition (C73T/R25X) located in the first coding exon (cmr1), a missense change (G482A/G161D) affecting a conserved glycine residue of the large intracellular loop (cmr2), and a frameshift mutation (C1388del/P463FS) resulting in a truncated peptide shortened by 92 C-terminal amino acids (cmr3). All three mutations lead to a highly consistent and similar phenotype in all cmr-affected dogs.23,24 Besides the genetic basis, cmr shares various clinical and pathologic manifestations with human bestrophinopathies and has been proposed as a relevant animal model for studying BEST1-associated phenotypes.23,24

In the present study, we have examined the mutation responsible for cmr1 (R25X), a premature termination codon of the canine BEST1, and compared the findings to the Y29X early stop mutation occurring in the human ortholog gene.25 Both mutations are positioned in the first mutational hotspot of the bestrophin-1 molecule. The ultimate goals of these studies were to characterize the bestrophin-1 null phenotype in a large, spontaneous animal model and to assess the molecular consequences of the nonsense mutation in cmr1-affected dogs and BEST1-mutated patients. In addition, the pathogenic effects of cmr2 missense substitution have been described and compared with the BEST1 null allele model. These evaluations provide a baseline for the future analysis of other deleterious sequence changes in the canine model with respective implications for human BEST1 phenotypes.

Materials and Methods

Animals and Tissue Processing

Dogs carrying the cmr1 mutation were used to produce either heterozygous or homozygous affected offspring. All animals underwent complete ophthalmic examinations, and fundus lesions were documented with fundus cameras (Genesis-D or RC-2 [Kowa Optimed, Inc., Torrance, CA]). For RNA or protein analysis, eyes were collected immediately after euthanatization at disease-relevant ages, dissected into RPE/choroid and retinal samples, frozen in liquid nitrogen, and stored at −70°C. For immunohistochemistry experiments, the enucleated globes were fixed in 4% paraformaldehyde, embedded in OCT medium, and processed as previously described.26 All procedures complied strictly with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

For RNA and protein analyses, HEK-293T cells were seeded in six-well plates (5 × 105) and grown overnight. Transient transfection was performed at 90% cell confluence with the wild-type (WT) or mutant expression plasmids and transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA) (1:5) in the presence of reduced-serum medium (OPTI-MEM I; Gibco Invitrogen, Grand Island, NY). Cell pellets were harvested 48 hours after transfection in ice-cold PBS. Each transfection experiment was carried out in triplicate, and cell pellets pooled from three wells were processed directly after harvesting. One day before transfection, MDCK cells used for immunohistochemistry experiments were re-plated on polycarbonate filters of 0.4-μm pore size (Transwell; Costar, Cambridge, MA) at 1 × 105 and allowed to grow for 3 to 4 days.27,28 The medium was changed every other day. After reaching 90% to 95% of confluence, monolayers were washed three times with reduced-serum medium (OPTI-MEM I; Gibco Invitrogen), and the complete growth media were replaced with EMEM supplemented with 5% FBS and absence of antibiotics. For each transfection sample, wild-type or mutant expression plasmid were diluted in reduced-serum medium (OPTI-MEM I; Gibco Invitrogen) and combined with transfection reagent (Lipofectamine 2000; Invitrogen; 1:6). After 40 minutes of incubation at room temperature, formed DNA-lipid complexes were applied to each of the experimental wells. Forty-eight hours later, confluent MDCK monolayers were subjected to immunohistochemical staining. All experiments were repeated at least three times. Two additional time points after transfection (24 hours and 72 hours) were evaluated as described (data not shown).

Protein expression level was determined by Western blot analysis according to the protocol described previously.23 Briefly, total protein was extracted from the RPE/choroids or cell pellets in 1 mL 1× Laemmli buffer, and 40 μg extracts were run on a 4% to 15% gradient polyacrylamide gel (Bio-Rad, Hercules, CA) before they were changed to a transfer membrane (Immobilon; Millipore, Billerica, MA). The membranes were probed with mouse monoclonal anti-BEST1 antibody (sc-32792; Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:100 dilution and were visualized with a rabbit anti-mouse IgG horseradish peroxidase secondary antibody (Zymed, Carlsbad, CA). The primary antibody used in the study (sc-32792; Santa Cruz Biotechnology), though raised against human bestrophin-1, specifically recognizes and binds to the canine C-terminal part of Best1 (Supplementary Fig. S2), as confirmed by a series of peptide competition assay experiments using the blocking peptide (sc-22027-P; Santa Cruz Biotechnology) supplied with the antibody. Additional anti-Best1 antibodies tested (ab14929, ab2182 [Abcam, Cambridge, MA]; sc-32792, sc-22027 [Santa Cruz Biotechnology[; ab14929, NB300–164 [Novus Biologicals, Littleton, CO]; Pab-125, E6–612) showed clear cross-reactivity with the canine-specific protein extracts; only ab14927 and ab14928 (Abcam, Cambridge, MA) did not cross-react with canine samples. ACTB was used as a reference protein and loading control (mouse monoclonal anti-actin, 1:10,000; Millipore, Temecula, CA). Each Western blot experiment was repeated at least three times. Relative band intensities were determined using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html).

Immunohistochemistry

Tissue sections were washed in 1× PBS/0.25% TX-100 for 5 minutes and were blocked for 1 hour (10% normal goat serum, 1× PBS/0.25% Triton X-100, 0.05% sodium azide). Overnight incubation with 1:400 anti-BEST1 mouse monoclonal antibody (sc-32792; Santa Cruz Biotechnology) at 4°C was followed by three washes with PBS for 5 minutes each and incubation with 1:200 goat anti-mouse 568 nm Alexa Fluor (Eugene, OR) for 1 hour at room temperature. After the washes, cell nuclei were stained with DAPI for 15 minutes. Sections were coverslipped with mounting medium (Gelvatol; Sigma) after a final wash and were examined by epifluorescence microscopy (Axioplan; Carl Zeiss, Thornwood, NY).

For all in vitro studies, MDCK cell monolayers were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.15% TX-100 in PBS for 10 minutes, and incubated with the primary antibody, mouse monoclonal anti-BEST1 (sc-32792, 1:400; Santa Cruz Biotechnology) overnight at 4°C. For double labeling, cells were incubated simultaneously with two antibodies, anti-BEST1 (as described) and Calnexin (1:500; ab13505; Abcam) or anti-BEST1 (as described) and anti-ZO-1 (1:100; ab59720; Abcam), followed by detection with Alexa Fluor 568 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit IgG, respectively, for 2 hours at room temperature. After DAPI staining and washes, filters (Transwell; Costar) were cut from the insets and mounted on microscope slides. Negative controls, nontransfected cells, mock-transfected cells, and samples without primary antibody were carried out in parallel. Each experiment was repeated at least three times.

Confocal Microscopy

Confocal images documenting the in vitro results were acquired on a tunable spectral confocal and multi-photon system (TCS-SP5; Leica Microsystems, Wetzlar, Germany) with an upright microscope (DM 6000 CFS; Leica Microsystems) through an HCX PL APO 40× (N.A. 1.25) or 60× (N.A. 1.40) oil-immersion objective. Alexa Fluor 488 and 568 were excited at 488 nm or 543 nm laser line, and emission was collected at 500 to 570 nm and 574 to 700 nm, respectively, in sequential scanning mode by the tunable internal detectors. DAPI was excited with multiphotons at 750 nm produced by a sapphire pulse laser (Chameleon Ultra II Ti; Coherent, Santa Clara, CA), and emission was collected at 380 to 500 nm. For some figures, Z stacks of a series of X-Y planes with a step size of 0.5 μm were captured.

Results

Fundus Phenotypes in cmr and BVMD

Canine multifocal retinopathy occurs in multiple dog breeds worldwide and manifests as bilateral multifocal areas of retinal elevations with subretinal deposits that have a tan-brown appearance (Figs. 1A1–A3). Both cmr1, caused by a C-to-T nonsense substitution, and cmr2, resulting from G-to-A missense change in cBEST1, result in a consistent phenotype that resembles the pathologic findings in BVMD patients (Figs. 1B1–1B3). BVMD is a clinically heterogeneous condition in which typical funduscopic changes with subretinal accumulation of egg yellow-like material predominantly affect the macular area (Fig. 1B1); in this case, the lesion is in the pseudohypopyon stage, which is also present in cmr (Fig. 1A2, inset23). However, multifocal distribution of lesions is observed in several families with autosomal dominant BVMD (Fig. 1B2) or compound heterozygous patients (Fig. 1B3). Peripheral lesions show discrepancies in number, size, and appearance and range from small and well-circumscribed yellow-white spots to large, diffuse lesions with irregular yellowish deposits, comparable to the central lesion (Figs. 1B1–1B3).

Fundus phenotypes in cmr and BVMD. (A1, A2) Fundus photographs of an 18-month-old English Mastiff with cmr1 and a 15-month-old Coton de Tulear with cmr2 (A3). In both animals, multiple subretinal lesions (arrowheads) are located in the superior tapetal region of the fundus. Note that the color of the lesions ranges from gray to tan-brown, influenced by the color of the overlying tapetum lucidum. (A2, inset) Fundus picture of 12-month-old Great Pyrenees with a pseudohypopyon-like lesion (arrow). (B1) Fundus appearance of a 47-year-old patient with unifocal BVMD lesion in the pseudohypopyon stage. The patient carries a Y227N mutation in the BEST1 gene and has a visual acuity of 20/80. (B2) Multifocal BVMD (K194_A195insV) in a 15-year-old patient with visual acuity of 20/100; arrowheads indicate some of the multiple retinal lesions scattered throughout the fundus periphery. (B3) Fundus photograph of a 10-year-old patient with multifocal vitelliform macular dystrophy (Y29X/R141H). Irregular vitelliform degeneration is present in the fovea (arrow). Note the rounded, vitelliform, yolk-like degeneration superiorly (arrowhead) and the more subtle, somewhat diffusely demarcated, vitelliform alterations along the superior vascular arcade. Fundus photographs provided courtesy of William Beltran (A1, A2), Bruce Grahn (A2, inset), Camiel Boon (B1, B2),11 and Patrik Schatz (B3).25

Figure 1.

Fundus phenotypes in cmr and BVMD. (A1, A2) Fundus photographs of an 18-month-old English Mastiff with cmr1 and a 15-month-old Coton de Tulear with cmr2 (A3). In both animals, multiple subretinal lesions (arrowheads) are located in the superior tapetal region of the fundus. Note that the color of the lesions ranges from gray to tan-brown, influenced by the color of the overlying tapetum lucidum. (A2, inset) Fundus picture of 12-month-old Great Pyrenees with a pseudohypopyon-like lesion (arrow). (B1) Fundus appearance of a 47-year-old patient with unifocal BVMD lesion in the pseudohypopyon stage. The patient carries a Y227N mutation in the BEST1 gene and has a visual acuity of 20/80. (B2) Multifocal BVMD (K194_A195insV) in a 15-year-old patient with visual acuity of 20/100; arrowheads indicate some of the multiple retinal lesions scattered throughout the fundus periphery. (B3) Fundus photograph of a 10-year-old patient with multifocal vitelliform macular dystrophy (Y29X/R141H). Irregular vitelliform degeneration is present in the fovea (arrow). Note the rounded, vitelliform, yolk-like degeneration superiorly (arrowhead) and the more subtle, somewhat diffusely demarcated, vitelliform alterations along the superior vascular arcade. Fundus photographs provided courtesy of William Beltran (A1, A2), Bruce Grahn (A2, inset), Camiel Boon (B1, B2),11 and Patrik Schatz (B3).25

To assess the molecular consequences of the cBEST1 early stop mutation (R25X), RPE tissues from wild-type, heterozygous (cmr1+/−), and homozygous affected (cmr1−/−) animals were evaluated at the mRNA and protein expression levels. Surprisingly, normal BEST1 mRNA expression levels were observed in the cmr1-affected animals and carriers compared with the wild-type tissue (Fig. 2A). These findings were verified by qRT-PCR, and no reduction of mRNA transcript levels was noted in either cmr1+/− or cmr1−/− RPE using both qRT-PCR probes located in the 5′-end or the 3′-end of the BEST1 cDNA sequence (Fig. 2B). On the other hand, Western blot analysis of RPE/choroid protein extracts and immunocytochemistry of retinal/RPE tissue sections clearly demonstrated a significant decrease in bestrophin-1 protein expression levels in R25X carriers (∼50% of the wild type) and absence of the gene product in homozygous mutant animals (Figs. 2C, 2D).

For the purpose of examining the molecular mechanism of the disease, an in vitro model system to recapitulate cmr1 has been developed. HEK-293T cells transiently transfected with cBEST1 wild-type or cmr1 mutant constructs revealed the same pattern of expression, accumulating an equivalent amount of mRNA transcript, and confirming the in vivo findings (Figs. 3A, 3B). To analyze bestrophin-1 protein expression, a series of transfection studies on the MDCK epithelial cell line, grown on permeable-filter supports mimicking the physiological polarized system, were performed (Fig. 4). As illustrated in Figures 3C (WT) and 4C and Supplementary Figure S3, cells transfected with the wild-type cBEST1 display robust, specific bestrophin-1 expression in the plasma membrane of the polarized cells. In contrast, this protein was not detectable in monolayers transfected with cmr1 mutant plasmid (Fig. 3C). Western blot results correlated with immunocytochemistry findings, confirming the absence of bestrophin-1 in a cmr1 in vitro model (Fig. 3D). In all cases, results were reproducible in at least three independent transfection experiments.

Cellular localization of WT bestrophin-1 in MDCK model system. Confocal images of polarized MDCK cell monolayer transfected with cBEST1 WT and grown on Transwell filters colabeled with DAPI (A, blue), tight junction marker ZO-1 (green), and bestrophin-1 (red). Z-stack images were obtained at 0.5-μm spacing, and the representative X-Y and X-Z (C, inset) sections demonstrate protein distributions, ZO-1 (arrows), and bestrophin-1 (arrowheads) at the apical surface (B) or the basal side (C) with a total of 4-μm difference between shown images. Note the colocalization of DAPI, a nuclear counterstain, with bestrophin-1 that can only be observed toward the basal side of the polarized epithelial cells (C). Scale bar, 5 μm.

Figure 4.

Cellular localization of WT bestrophin-1 in MDCK model system. Confocal images of polarized MDCK cell monolayer transfected with cBEST1 WT and grown on Transwell filters colabeled with DAPI (A, blue), tight junction marker ZO-1 (green), and bestrophin-1 (red). Z-stack images were obtained at 0.5-μm spacing, and the representative X-Y and X-Z (C, inset) sections demonstrate protein distributions, ZO-1 (arrows), and bestrophin-1 (arrowheads) at the apical surface (B) or the basal side (C) with a total of 4-μm difference between shown images. Note the colocalization of DAPI, a nuclear counterstain, with bestrophin-1 that can only be observed toward the basal side of the polarized epithelial cells (C). Scale bar, 5 μm.

To compare the molecular consequences of bestrophin-1 null phenotype (cmr1) with the spontaneous cmr2 missense-mutated cBEST1 model, a series of comparable in vitro evaluations were performed. The G482A/G161D missense mutation changes a highly evolutionarily conserved glycine residue to the polar and negatively charged aspartic acid and results in a phenotype indistinguishable from cmr1 (Figs. 1A1–1A323). At the mRNA expression level, the cmr2 transition behaves similarly to the wild type, suggesting that mRNA stability is not altered by the guanine to adenine substitution in exon 5 of cBEST1 (Figs. 3A, 3B). Even though Western blot analysis results do not indicate any abnormalities of the cmr2-affected gene product (Fig. 3D), in vitro data on the polarized model system demonstrate mislocalization of bestrophin-1 to the perinuclear space of the cells when transiently transfected with the G482A mutant construct (Fig. 3C). Detailed analysis revealed colocalization of cmr2 mutant protein with Calnexin, an endoplasmic reticulum marker (Fig. 3E).

Comparison of Canine R25X and Human Y29X Mutations

To further investigate the nonsense-mediated mRNA decay insensitivity observed with cmr1, we examined a comparable early stop mutation (C87G/Y29X) of the hBEST1 gene. Similar to canine C73T, the C87G nonsense substitution was positioned in close proximity to the AUG-start codon, introducing a premature stop signal within the first coding exon of the human ortholog (Supplementary Fig. S1). An hBEST1 construct carrying the C87G change was created by site-directed mutagenesis and evaluated in the in vitro model system under the same conditions described for cmr1.

The results, summarized in Figure 5, clearly demonstrate that the Y29X mutation behaves in the same manner as cmr1. No variation in abundance of the mRNA expression level in C87G mutants compared with the wild-type transcript was observed when amplified by standard RT-PCR or qRT-PCR (Figs. 5A, 5B). The hypotheses of alternative start codon use, exon skipping concept, or involvement in alternative gene-splicing machinery were rejected for both canine and human BEST1. All PCR products spanning the region with mutations of interest corresponded to the expected sizes and were analyzed with at least two different primer pairs and confirmed by direct sequencing (data not shown).

Bestrophinopathies are a group of inherited retinal disorders primarily caused by point mutations scattered throughout the entire BEST1 gene. Because of largely overlapping clinical manifestations among the BEST1-defined phenotypes, frequent incomplete penetrance, and, most important, markedly variable expressivity, attempts to elucidate the genotype-phenotype relationship within this group of retinopathies are difficult.10,30–32 Remarkable progress, however, has been made in establishing a link between mutated bestrophin-1 residues and the anion channel malfunction, which significantly improved our understanding of the BEST1 disease mechanism.6,13,19,22 Most frequently, diminished or absent Cl− current, altered anion permeability, or defective membrane integration in association with BEST1 nucleotide substitutions have been reported (see Refs. 7, 17 for reviews). Despite the indisputable advancements made in the elucidation of bestrophin-1 pathogenic effects, the molecular behavior of mutant transcripts and the expression of altered bestrophin-1 protein and its impact on intracellular trafficking were either not examined at all or were studied in only few cases.6,7,17 As a result, molecular and functional consequences of BEST1 mutations remain largely unexplored; thus, even mutations identified in early childhood do not translate to a clear prognosis. It seems, therefore, that progress in the dissection of the complex BEST1 genotype-phenotype relationship and the establishment of a strong correlation between a particular mutation and bestrophin-1 malfunction is conditioned by recapitulating the disease phenotype in a reliable animal model.

The domestic dog represents a model that shares not only nearly half its known hereditary disorders with humans but also environmental factors and common habits. These features posit that this species is an excellent model organism for studying both monogenic and complex genetic traits.33,34 In the past decade, use of canines as a natural model for studying inherited blinding disorders has driven this field toward the discovery of novel disease-causing mutations and substantially deepened our understanding of retinal degenerative mechanisms (see Ref. 35 for review). Moreover, the dog model has primarily contributed to the development of successful vision preservation or restoration therapies that are applicable to humans.36–40

Canine multifocal retinopathy is the only spontaneous large animal model available for studying the complexity of BEST1-associated phenotypes.23,24 Besides having mutations in the same ortholog, cmr shares numerous phenotypic features with human bestrophinopathies in which multifocal along with focal-macular lesions have been documented.5,9,11,25 Characterization of the unique cmr1 animal model in the present study verified a BEST1 null phenotype in which C73T/R25X nonsense transition encodes for a truncated protein not detectable in the homozygous affected RPE or the cmr1-induced in vitro model. This results in the complete loss of bestrophin-1 function.

As described previously, the cmr1 phenotype is indistinguishable from cmr2, the second naturally occurring canine BEST1 model caused by the missense mutation G482A.23 The G-to-A substitution at the beginning of exon 5 of cBEST1 locates the mutated amino acid residue in the large intracellular loop of the bestrophin-1 protein (106–230aa; Supplementary Fig. S1), a region that is thus far mainly uncharacterized.8,17 To date, 20 disease-causing mutations in hBEST1 are reported within this region, but their molecular or functional consequences have not been examined.7,17

In the cmr2 model, the small, neutral and non-hydrophilic glycine residue at position 161 is replaced by a much larger, highly hydrophilic and negatively charged aspartic acid. We speculated that this drastic amino acid change may cause defective intracellular trafficking because of the incorrect folding of the mutant protein that cannot pass the endoplasmic reticulum quality control system and is subjected to degradation.41 The fact that cmr2-affected bestrophin-1 protein can only be found in the endoplasmic reticulum, but not in the plasma membrane, supports our hypothesis and provides an elegant explanation of why two distinct BEST1 mutations result in an indistinguishable phenotype. Both encode for the altered gene product that presumably results in a nonfunctional anion channel.

Nonsense mutations introducing premature termination codons (PTCs), located in the 5′ end of the gene coding sequence, are commonly considered as null allelic mutations resulting in the production of a truncated, usually inactive, protein.42 In mammalian cells, a highly specialized surveillance pathway, termed nonsense-mediated mRNA decay (NMD), is responsible for the detection and degradation of all aberrant transcripts generated by PTCs. The NMD apparatus recognizes termination codons as premature if they are located >50 to 54 nucleotides 5′ to the final exon-exon junction (EEJ) and subjects the affected mRNAs to rapid degradation.42,43 However, the 50- to 54-nt boundary rule has been seriously challenged in a few studies describing gene transcripts carrying PTCs and accumulating expression levels similar to the wild type, thus resistant to the NMD machinery.44–47 In our studies, we observed the same phenomenon of NMD insensitivity occurring with the canine C73T and the comparable human C87G mutation. In both cases, the generated premature stop codons are located >50 bp upstream of the last EEJ and, hence, should represent an adequate target for NMD.

Although the molecular machinery of NMD avoidance remains largely unknown, Inacio et al.45 proposed an AUG proximity effect as an underlying mechanism for mRNA molecules that do not undergo NMD but that contain PTCs located in the vicinity of the AUG start codon. The AUG proximity effect represents a novel parameter in the NMD complex response, blunting mRNA destabilization through the presence of a nonsense mutation at the 5′ terminus of the open reading frame, which appears to dominate the 50- to 54-nt boundary rule.45 Our study supports the AUG proximity effect as a molecular mechanism underlying the cmr1 and Y29X BEST1 mutations and provides evidence that this NMD parameter is neither tissue nor species specific. Such factors as NMD or its resistance may contribute to the modulation of the phenotypic outcomes of the disease, originated by nonsense mutation and the variable expressivity observed in BEST1-related phenotypes. To our knowledge, this is the first report demonstrating the NMD resistance effect in conjunction with a retinal disease–associated gene.

The assessment of molecular and functional consequences of cmr1 and cmr2, two canine models recapitulating human bestrophinopathy phenotypes, constitute the first step toward the elucidation of genotype-phenotype interactions of bestrophinopathies and indicate the importance of the canine model for studying the complexity of the disease mechanism and the development of future therapeutic approaches.

Fundus phenotypes in cmr and BVMD. (A1, A2) Fundus photographs of an 18-month-old English Mastiff with cmr1 and a 15-month-old Coton de Tulear with cmr2 (A3). In both animals, multiple subretinal lesions (arrowheads) are located in the superior tapetal region of the fundus. Note that the color of the lesions ranges from gray to tan-brown, influenced by the color of the overlying tapetum lucidum. (A2, inset) Fundus picture of 12-month-old Great Pyrenees with a pseudohypopyon-like lesion (arrow). (B1) Fundus appearance of a 47-year-old patient with unifocal BVMD lesion in the pseudohypopyon stage. The patient carries a Y227N mutation in the BEST1 gene and has a visual acuity of 20/80. (B2) Multifocal BVMD (K194_A195insV) in a 15-year-old patient with visual acuity of 20/100; arrowheads indicate some of the multiple retinal lesions scattered throughout the fundus periphery. (B3) Fundus photograph of a 10-year-old patient with multifocal vitelliform macular dystrophy (Y29X/R141H). Irregular vitelliform degeneration is present in the fovea (arrow). Note the rounded, vitelliform, yolk-like degeneration superiorly (arrowhead) and the more subtle, somewhat diffusely demarcated, vitelliform alterations along the superior vascular arcade. Fundus photographs provided courtesy of William Beltran (A1, A2), Bruce Grahn (A2, inset), Camiel Boon (B1, B2),11 and Patrik Schatz (B3).25

Figure 1.

Fundus phenotypes in cmr and BVMD. (A1, A2) Fundus photographs of an 18-month-old English Mastiff with cmr1 and a 15-month-old Coton de Tulear with cmr2 (A3). In both animals, multiple subretinal lesions (arrowheads) are located in the superior tapetal region of the fundus. Note that the color of the lesions ranges from gray to tan-brown, influenced by the color of the overlying tapetum lucidum. (A2, inset) Fundus picture of 12-month-old Great Pyrenees with a pseudohypopyon-like lesion (arrow). (B1) Fundus appearance of a 47-year-old patient with unifocal BVMD lesion in the pseudohypopyon stage. The patient carries a Y227N mutation in the BEST1 gene and has a visual acuity of 20/80. (B2) Multifocal BVMD (K194_A195insV) in a 15-year-old patient with visual acuity of 20/100; arrowheads indicate some of the multiple retinal lesions scattered throughout the fundus periphery. (B3) Fundus photograph of a 10-year-old patient with multifocal vitelliform macular dystrophy (Y29X/R141H). Irregular vitelliform degeneration is present in the fovea (arrow). Note the rounded, vitelliform, yolk-like degeneration superiorly (arrowhead) and the more subtle, somewhat diffusely demarcated, vitelliform alterations along the superior vascular arcade. Fundus photographs provided courtesy of William Beltran (A1, A2), Bruce Grahn (A2, inset), Camiel Boon (B1, B2),11 and Patrik Schatz (B3).25

Cellular localization of WT bestrophin-1 in MDCK model system. Confocal images of polarized MDCK cell monolayer transfected with cBEST1 WT and grown on Transwell filters colabeled with DAPI (A, blue), tight junction marker ZO-1 (green), and bestrophin-1 (red). Z-stack images were obtained at 0.5-μm spacing, and the representative X-Y and X-Z (C, inset) sections demonstrate protein distributions, ZO-1 (arrows), and bestrophin-1 (arrowheads) at the apical surface (B) or the basal side (C) with a total of 4-μm difference between shown images. Note the colocalization of DAPI, a nuclear counterstain, with bestrophin-1 that can only be observed toward the basal side of the polarized epithelial cells (C). Scale bar, 5 μm.

Figure 4.

Cellular localization of WT bestrophin-1 in MDCK model system. Confocal images of polarized MDCK cell monolayer transfected with cBEST1 WT and grown on Transwell filters colabeled with DAPI (A, blue), tight junction marker ZO-1 (green), and bestrophin-1 (red). Z-stack images were obtained at 0.5-μm spacing, and the representative X-Y and X-Z (C, inset) sections demonstrate protein distributions, ZO-1 (arrows), and bestrophin-1 (arrowheads) at the apical surface (B) or the basal side (C) with a total of 4-μm difference between shown images. Note the colocalization of DAPI, a nuclear counterstain, with bestrophin-1 that can only be observed toward the basal side of the polarized epithelial cells (C). Scale bar, 5 μm.